Human granulocytic anaplasmosis (HGA) is one of most prevalent life-threatening tick-borne zoonoses in North America. This disease is caused by Anaplasma phagocytophilum, a Gram-negative obligatory intracellular bacterium. A. phagocytophilum replicates within membrane-bound inclusions in human polymorphonuclear leukocytes (PMNs), which are primary host defensive cells, by subverting innate immune responses and sequestering nutrients. The present proposal aims to elucidate three essential host subversive events: A. phagocytophilum cholesterol acquisition, autophagosome formation, and extracellular exit. Our first hypothesis is that A. phagocytophilum hijacks LDL-derived cholesterol vesicular transport to divert cholesterol into its inclusion. Our second hypothesis is that A. phagocytophilum subverts autophagy by activating class III phosphatidylinositol 3-kinase (PI3KC3) and preventing autophagosome maturation. Our third hypothesis is that A. phagocytophilum can exit host cells by exploiting the PMN degranulation regulatory pathway. Therefore, to test these three hypotheses, our specific research aims are to: 1. study the trafficking various proteins known to be involved in cholesterol vesicular transport, particularly from NPC1 vesicles to the transGolgi network, and lipid raft-associated proteins, flotillins to A. phagocytophilum inclusions, and requirement of these proteins for A. phagocytophilum infection, and identify bacterial proteins involved in this process. 2. examine localization and activities of proteins involved in autophagy induction and maturation on A. phagocytophilum inclusions, and characterize the nature of autophagosome-like membrane vesicles induced by a type IV secretion substrate. 3. study the localization and regulation of Rab and SNARE proteins (Rab27a, JFC1, Vamp2, and syntaxin 4) that are involved in secretory granule exocytosis in PMNs on A. phagocytophilum inclusions and identify bacterial proteins potentially recruiting the SNARE and Rab proteins to A. phagocytophilum inclusions. The data to be obtained from this study will provide a breakthrough in new understanding on mechanisms of host cell subversion by obligatory intracellular bacteria and functions of inclusions. The results may point to a potential target for new treatment and prevention of HGA. Our study will enhance understanding of the LDLR cholesterol vesicular transport pathway, introduce a new paradigm for regulation of subcellular sites of autophagosome formation and maturation, and provide new insights into regulation of secretory granules.